Activation and substrate specificity of the human P4-ATPase ATP8B1

Asymmetric distribution of phospholipids in eukaryotic membranes is essential for cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in creating and maintaining this asymmetry through active transport of phospholipids from the exoplasmic to the cytosolic leaflet. Here, we present a total of nine cryo-electron microscopy structures of the human flippase ATP8B1-CDC50A complex at 2.4 to 3.1 Å overall resolution, along with functional and computational studies, addressing the autophosphorylation steps from ATP, substrate recognition and occlusion, as well as a phosphoinositide binding site. We find that the P4-ATPase transport site is occupied by water upon phosphorylation from ATP. Additionally, we identify two different autoinhibited states, a closed and an outward-open conformation. Furthermore, we identify and characterize the PI(3,4,5)P3 binding site of ATP8B1 in an electropositive pocket between transmembrane segments 5, 7, 8, and 10. Our study also highlights the structural basis of a broad lipid specificity of ATP8B1 and adds phosphatidylinositol as a transport substrate for ATP8B1. We report a critical role of the sn-2 ester bond of glycerophospholipids in substrate recognition by ATP8B1 through conserved S403. These findings provide fundamental insights into ATP8B1 catalytic cycle and regulation, and substrate recognition in P4-ATPases.


Supplementary Discussion
Is there a second magnesium ion in the AMPPCP binding pocket?
In our cryo-EM map of the ATP8B1-CDC50A complex trapped in the E1-ATP conformation with AMPPCP, a density could be observed at proximity of the beta-phosphate of the AMPPCP molecule (see picture below).
Close-up view of the electron density map (4.2σ) of AMPPCP and neighboring residues of ATP8B1, highlighting the presence of an unassigned extra density near the beta phosphate.

Supplementary Figure 2 -b
Biochemical characterization of the purified ATP8B1-CDC50A complex.a) SDS-PAGE of the purified ATP8B1-CDC50A complexes (WT full-length and C-terminally truncated) used for cryo-EM and ATPase activity measurements in this study.b) Size exclusion chromatography (Superose 6 increase 10/300) elution profiles from the last purification step of the WT full-length and C-terminally truncated ATP8B1-CDC50A complexes.a Patch motion correction and patch CTF estimation (11,168 micrographs) Selection of good micrographs (10,722 micrographs) Template based picking (ref: EMDB-13711) (3,571,944 particles) Particle extraction 3,571,944 particles, box size: 384 px, cropped to 96 px Selection of junk 2D class Selection of good 2D class Ab initio (4 volumes) Heterogeneous refinement 1 (HR1) with all particles

Final
stack : 608,138 good particles re-extraction, box size: 384 px, cropped to 240 px Heterogeneous refinement 4 with the good particles from HR3 (640,662 particles) (1 good vs 4 junks) 3D variability and clustering (8 class) Continuous motion of the A-and N-domains observed Selection of 3 similar clusters (226,859 particles)Non-uniform refinement (+ local CTF refine)

bSupplementary 3 Supplementary
18 -Comparison of the canonical transport site in different Ptype ATPases in E1 or E1-ATP states.The comparison of the position of the water molecules observed in P4-and P5-ATPAse (ATP8B1 and ATP13A2) is similar to the position observed for cations in P1-and P2-ATPases (CopA, ATP2A1 and ATP1A1).Supplementary Figure 19 -Comparison with the previously published structures of ATP8B1-CDC50A autoinhibited states.a) ATP8B1-CDC50A in the E2P autoinhibited "closed" conformation (left) is similar to the previously published structure by Dieudonné et al., 2022.ATP8B1-CDC50A in the E2P autoinhibited "open" conformation (right) is similar to the previously published structure by Cheng et al., 2022.b) Structural alignment of the three E2P conformations described in this study.E2P autoinhibited "open" and the E2P active show a similar TM1-2 open conformation.a This study: E2P autoinhibited "closed" E2P autoinhibited "open" E2P active E2P autoinhibited "closed" (this study) vs 7PY4 (Dieudonné et al.,2022) E2P autoinhibited "open" (this study) vs 7VGI (Cheng et al.,2022) TM1-2 RMSD : 2.39 Å TM1-2 RMSD : 1.40 Å TM1-2 RMSD "closed" vs "open" : 5.77 Å "closed" vs "active": 5.92 Å "open" vs "active": 1.15 Å Figure 20 -Comparison of the lipid groove of autoinhibited and active P4-ATPases in E2P state.The structure of the autoinhibited P4-ATPases in the E2P state display a close lipid entry groove while active proteins are open and filled with lipid substrate.comparison of the different phosphoinositide binding site observed in two P4-ATPases, ATP8B1 and Drs2p and in the P5-ATPase ATP13A2.The comparison of the phosphoinositide binding site observed in other P-type ATPases (Drs2 and ATP13A2) reveals that all phosphoinositides bind a similar region of the protein located in a cavity formed by TM7 and TM10 (and TM5 for P4-ATPases) filled with positively charged residues.-PI(3,4,5)P 3 binding site in other E2P conformations and complementary analysis of the MD data.a) PI(3,4,5)P 3 binding site and the lipid associated EM density in the three E2P states of ATP8B1.b) Comparison of PI(3,4,5)P 3 position before (blue) and after (orange) the initial MD equilibration step.c) All Clusters of inositol ring coordinates from the last 200 ns of each trajectory with Daura's algorithm for PI(3,4,5)P 3 (blue) and PI (purple).d) Hydration of the lipid binding site.The average number of water molecules present in the lipid binding site for the PI(3,4,5)P 3 -bound system (blue) and the PI-bound (purple) over the last 200 ns of the simulations.The shaded area indicates the standard deviation over the five replicates of each system.e) K + ions surrounding the lipid headgroup.The average number of K + ions present within 6 Å of the PI(3,4,5)P 3 and the PI (purple) headgroup (blue) over the last 200 ns of the simulations.The shaded area indicates the standard deviation over the five replicates of each system.ATP8B1 E2P active ATP11C E2P (7BSU) Dnf1p E2P (7DRX) Drs2p E2P active (7PEM) Supplementary Figure 23 -Comparison of the lipid binding site in P4-ATPases in E2P active conformation.Exoplasmic view of the transport lipid binding site of different P4-ATPase in E2P active conformation.The surface around the transport lipid is shown as electrostatic surface.up view of the transport lipid binding site in the E2P active and E2┅Pi (lipid occluded) conformation and their associated electron map.a) E2P active conformation (contour map: 5.7σ), b) E2┅Pi (PI) conformation (contour map: 4.34σ step 2), c) E2┅Pi (PC) conformation (contour map: 3.3σ), d) E2┅Pi (PS) conformation (contour map: 4.3σ).In b, c and d, arrows indicated continuous densities observed between the possible different rotamers of the S403 and the phosphate of (blue arrow) and the sn2ester bond of the lipid (black arrow).binding site in the different ATP8B1-CDC50A conformations reported in this study and its putative link with the lipid transport site.a) Comparison of the PI(3,4,5)P 3 binding site (red asterisk) conformation in the different structures presented in this study.b) Close-up view of the PI(3,4,5)P 3 binding site and the transport lipid binding (PC) site in the E2P active conformation.PI(3,4,5)P 3 tightly interacts with R952 on TM5 adjacent to the lipid transport site.For clarity purposes, only TM4 and TM5 are shown.Figure 26 -TM1-2 orientation in ATP8B1 and ATP11C in E2P active conformation.ATP8B1 TM1-2 are shown in brown, ATP11C (PDB: 7BSU) TM1-2 are shown in blue.The lipid recognition motif (PISL) of TM4 is shown in purple.CDC50A (from the ATP8B1-CDC50A complex) is shown in pink.alignment of TM1-4 of ATP8B1 and PS-specific human P4-ATPases.a) ATP8B1 in the E2P active PC bound conformation with a close-up view of TM1-4 organization close to the lipid binding site.TM1, TM2, TM3 and TM4 are blue, turquoise, yellow and red, respectively.The non-conserved residues between ATP8B1 and PSspecific human P4-ATPases are shown as sticks.b) Sequence alignment of TM1-4 of ATP8B1 and PS-specific human P4-ATPases, colors as in a).(Uniprot ID: hATP8B1(O43520); hATP8A1 (Q9Y2Q0); hATP8A2 (Q9NTI2); hATP11A (P98196); hATP11B (Q9Y2G3); hATP11C (Q8NB49)).

Table 1 -Data collection and refinement statistics
5,2.However, to our knowledge, it has never been observed or modeled for P-type ATPase trapped with AMPPCP.In a recent Interestingly, during their MD simulation, Mateeva et al. also noticed that potassium ions (the only monovalent cation included in their study) can bind at the same position.To gain a better understanding of which element is responsible for the extra density observed in our map, we compared it with all available cryo-EM maps of P-type ATPase trapped with AMPPCP.We also examined the buffer composition of the sample used for grid freezing (see table below).Unfortunately, we could not find any clear correlation between the cations present in the samples and the presence of the extra density in the published cryo-EM maps.This suggests that different ions such as sodium (Na + ), potassium (K + ), or magnesium (Mg 2+ ) may partially occupy the site, in line with the weaker intensity of this density in all EM maps compared to the other magnesium site (site I).For consistency, we chose not to model any ion into this density.Lastly, we did not model a second magnesium ion in our E1P-ADP model due to the uncertainty in our cryo-EM map in this region.NaOH pH 7.5, 150 mM NaCl,5mM MgCl 2 , and 1 mM DTT / 2 mM AMPPCP Mg 2+ , Na + YES 9 Xu et al., 2022 7WHW Dnf2 20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , and 1 mM DTT / 2 mM AMPPCP Mg 2+ , Na + NO 9 Xu et al., 2022 NaOH pH 7.0, 100 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM DTT, and 0.1 mg.mL -1 LMNG / 1 mM AMPPCP Mg 2+ ,Ca 2+ ,K + YES 12 Zhang et al., 2020 6LN5 Serca2b 50 mM Hepes-NaOH pH 7.0, 100 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM DTT, and 0.01% (w/v) LMNG / 1 mM AMPPCP Na + Mg 2+ ,Ca 2+ ,K + YES 12 Zhang et al., 2020 6LN6 Serca2b 50 mM Hepes-NaOH pH 7.0, 100 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM DTT, and 0.01% (w/v) LMNG / 1 mM AMPPCP Na + , Mg 2+ ,Ca 2+ ,K + YES 12 Zhang et al., 2020 6LN7 Serca2b 50 mM Hepes-NaOH pH 7.0, 100 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM DTT, and 0.01% (w/v) LMNG / 1 mM AMPPCP Mg 2+ ,Ca 2+ ,K + YES 12 Zhang et al., 2020 a position similar to our observed extra density, plays a critical role in the transfer of the gamma-phosphate of ATP to the catalytic aspartate of P-type ATPase.